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You could try the approach described by Mashanov and Molloy in j biol chem 2003, effectively varying the laser power, which changes the rate of bleaching, but also tells you if there is unbinding. See paper for more explanation


Michelle

On 10 May 2013, at 16:19, "Jeff Spector" <[log in to unmask]> wrote:

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> 
> Thanks for the references. I've looked at a lot of that type of literature.
> The issue I am having is that we have a nice TIRF image of single molecules
> landing on our substrate and then disappearing. I need to know if they are
> dissociating from the substrate or bleaching.  I see a lot of molecule on
> glass in my samples, and I guess I could measure their bleaching curves,
> but how do I know they aren't dissociating from the glass? I guess I could
> get a distribution of glass bound molecules and measure their bleaching
> curves to get an average bleaching time, and then compare that with the
> dwell times I measure on the sample, and as long as the dwell times are
> shorter than the average on-glass bleaching time then I can use this
> measurement, and if not then I need to adjust my framerate or laser power.
> Is this the correct way to do this sort of  'dwell time' analysis?  We are
> not doing FRET and we don't really need to count the number of molecules in
> a given fluorescent spot, we really just want to measure the "on" and "off"
> rates. Anyone else have some literature I can look at, I seem to be having
> a hard time finding anything..
> thanks...
> -jeff
> 
> 
> On Thu, May 9, 2013 at 11:44 PM, John Oreopoulos <
> [log in to unmask]> wrote:
> 
>> *****
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>> 
>> Jeff,
>> 
>> Take a look at these articles. Might be some help here:
>> 
>> Coffman, V.C. and J.Q. Wu, Counting protein molecules using quantitative
>> fluorescence microscopy. Trends in Biochemical Sciences, 2012. 37(11): p.
>> 499-506.
>> 
>> McGuire, H., et al., Automating single subunit counting of membrane
>> proteins in mammalian cells. Journal of Biological Chemistry, 2012.
>> 287(43): p. 35912-35921.
>> 
>> John Oreopoulos
>> 
>> 
>> On 2013-05-09, at 10:07 PM, Jeff Spector wrote:
>> 
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>>> 
>>> Greetings,
>>> this may be somewhat out of place in this listserv but I know a lot of
>> good
>>> microscopists are on this list.
>>> I'm using a single molecule assay to try and measure the binding
>> kinetics of an
>>> enzyme. Essentially, I can see them land on my substrate and then
>> disappear. I
>>> would like to measure the on and off rates but am unsure as to how to
>> include
>>> the effects of photobleaching. I have seen a lot of papers where people
>> "correct"
>>> for this but don't explain how. Could someone on this list point me
>> towards some
>>> literature that might help me out?
>>> thanks...
>>